The SPIM technique (single plane illumination microscopy), in which illumination of the sample is carried out in layers, permits an acquisition of image data that is faster and gentler on the sample than point-by-point scanning of a sample, for example. A known application of SPIM technology is the field of fluorescence microscopy, in which fluorophores in the sample are excited by laser light. In the known SPIM technology, an excitation takes place in this case in a layer of a sample illuminated by an illumination light sheet (also termed a “light strip”). To generate a SPIM image, the sample is illuminated by the sheet of light, while the observation of the sample layer illuminated in this way takes place in a vertical direction by detection of the fluorescent or scattered light. From the successively obtained images of different sample layers, in particular from a stack of images of sample layers parallel to one another, a three-dimensional representation of the sample can be generated.
A microscope that works according to the SPIM method is described in DE 102 57 423 A1. With this microscope a sample is illuminated by a thin light strip, while the observation takes place perpendicular to the plane of the illuminating light strip. The illumination and the detection take place by way of two separate optical beam paths, each having separate optical systems, in particular having two separate lenses perpendicular to one another. The light strip is generated by an illumination lens and a cylindrical optical system upstream of this. To record the image, the sample is moved through the light strip, which is stationary relative to the detector, in order to record fluorescent and/or scattered light in layers using a two-dimensional detector. The layer image data obtained in this way can then be assembled into a dataset corresponding to a three-dimensional image of the sample.
An arrangement is disclosed in DE 10 2004 034 957 A1 for the microscopic observation of a sample via a microscope lens, in the housing of which, apart from the lens optical system, additional light guides are provided for the illumination light. The illumination light runs in this case initially parallel to the optical axis of the lens inside the light guides and then encounters an annular reflector having a small aperture mounted on the lens housing, which aperture focuses the illumination light with the aid of additional imaging elements perpendicular to the optical axis of the microscope lens and thus perpendicular to the observation direction into the sample. The illumination of the sample takes place two-dimensionally according to the SPIM principle here too. One problem in particular with this microscope is having to position the sample spatially inside the annular reflector.
An arrangement for illuminating a sample in SPIM microscopy is also disclosed in DE 20 2011 110 077 U1. The arrangement includes a light source for generating a light beam, means for generating a light strip from the light beam, and at least one lens, which has an optical system that is formed and intended to supply detection light emanating from the sample directly or indirectly to a detector. The arrangement also includes a deflection means downstream of the optical system of the lens for deflecting the light strip.